Oral immunogens have the potential to be some of the most versatile, cost effective and potent vaccines for stimulating mucosal immune responses. However fundamental principles for the design and use of effective oral, multivalent, subunit vaccines are developing. For this proposal, a multidisciplinary team will investigate in depth the immune response against two different subunit vaccine candidates administered orally in a murine model. Vaccine candidates to be used in this proposal are derived from microbes which cause significant diarrheal disease in humans; i.e. the heat labile toxin of E. coli (LT-B) and the capsid protein of Norwalk virus. The form of antigen used as well as the scheme for oral immunization will be varied between immunogens given 1) as recombinant protein, 2) associated with liposomes, 3) as Salmonella constructs, or 4) expressed in plants. Both the inductive and effector phases of the mucosal immune response will be evaluated after primary immunizations and after subsequent secondary immunizations. To evaluate the immune response, advanced technologies will be employed. Specifically, sensitive quantitative competitive-reverse transcribed-polymerase chain reactions (QC-RT-PCR) will be used to define the primary and secondary inductive phases of the cytokine response in the Peyer's patches, intestines, mesenteric lymph nodes and spleen of orally immunized animals. Furthermore the primary and secondary effector phases of the immune response in the same tissues will be detailed using l) QC-RT-PCR for cytokine mRNAs, 2) ELISpot analyses for cytokine secretion, 3) ELISAs to define antigen specific antibody secretion, 4) antibody neutralization assays for LT-B and Norwalk virus, 5) ELISPot analyses for antigen specific antibody secretion, and 6) in vitro antigen-induced T helper lymphocyte activation. Initially, the same antigen preparation (i.e. homologous immunizations) will be given orally to stimulate secondary immune responses. These studies will not only establish a standard level of responsiveness for each immunization scheme, but will permit comparison to identify the "optimal" methods for stimulating the appropriate response for each antigen. Once this has been accomplished, it will be possible to investigate strategies for combinatorial oral immunizations using both antigens. These studies will utilize the "best" immunization strategies for both antigens given to a single animal, and the immune response will be evaluated as described above. Using a similar strategy, the memory immune response will be investigated to determine the potential effects of immunological interference, antigenic competition, or as yet other undefined problems or benefits associated with giving multiple oral immunizations. Ultimately, these studies will address fundamental questions relative to the combinations and immunization schemes which are optimal for the development of multivalent oral vaccines.